GSM6732892: Neocortical lesion, TSC, 4259; Homo sapiens; RNA-Seq
A fresh-frozen brain tissue (~50 mg) was placed into a glass dounce homogenizer containing 1 ml cold lysis buffer (0.05% (v/v) NP-40, 10 mM Tris (pH 7.4), 3 mM MgCl2, 10 mM NaCl) and dounce 10 times with a loose pestle and following 10 times with a tight pestle. The homogenate was incubated for 10 min in RT. 9 ml of wash buffer (1% BSA in 1X PBS) was added to the homogenate and filtered by a 30 um cell strainer. The strained homogenate was spun down in 500 g to remove the supernatant. The pellet was resuspended with 5 ml of wash buffer. Straining and spinning down steps were performed once more, and the pellet was resuspended into 500 ul of wash buffer. 10 ul of nuclei resuspension was mixed with counting solution (0.02% Tween 20, 0.1ug/ml DAPI, 1% BSA in 1X PBS) and nuclei density was measured by manual nuclei counting using DAPI signal. The resuspension was diluted by wash buffer to make the desired concentration (800~1000 nuclei/ul). Maximum 2 samples were pooled together targeting 10000 nuclei per reaction. Gel beads emulsion (GEM) generation, cDNA, and sequencing library constructions were performed in accordance with instructions in the Chromium Single Cell 3' Reagent Kits User Guide (v3.1). A library pool was sequenced with 800 million read pairs using NovaSeq 6000.